The optimum conditions for production of human gamma interferon (HuIFN gamma) by protein A from Staphylococcus aureus (SpA) were determined. Dose-response experiments showed donor-to-donor variability in the concentration of SpA required for maximum HuIFN gamma production. Although variation was observed, SpA at 50 micrograms/microliter was at or near peak HuIFN gamma values for all donors tested. Kinetic studies showed that peak HuIFN gamma titers were obtained 24 hours after SpA stimulation. Further studies revealed that lymphocyte concentrations of 2-5 by 10 to the 6th power/microliter provided maximum HiuIFN gamma yields. Moreover, HuIFN gamma-containing supernatants could be harvested for 3 successive days by removing supernatants and adding fresh medium to stimulated lymphocytes, thus providing substantially increased HuIFN gamma yields from stimulated lymphocytes. Preliminary cell fractionation experiments were performed to determine the lymphocyte subpopulation that produces HuIFN gamma after SpA stimulation. These data suggest that the HuIFN gamma-producing cell is a nonadherent lymphocyte that lacks high affinity receptors for sheep erythrocytes and expresses Fc receptors. Additional studies are in progress to confirm these observations.